There is no disease prevention approach more broadly powerful than vaccination. Unfortunately, few well-defined vaccines against bacterial pathogens are available today. Identification of bacterial surface components that would make effective vaccine targets is clearly a public health priority. One such target is the type III secretion system (T3SS) used by many Gram-negative pathogens to deliver virulence proteins to eukaryotic cells for altering their normal functions. T3SSs are surface exposed supramolecular machines composed of two main structural features: a) a complex basal body that spans the inner and outer bacterial membranes; and b) a surface exposed needle composed of multiple copies of a single protein and capped with a tip complex that is needed to sense host cell contact to facilitate efficient insertion of bacterial proteins into the host cell membrane. For Shigella flexneri, the causative agent bacillary dysentery, the needle monomer is MxiH and the tip complex is composed of the invasion plasmid antigen IpaD. Both of these proteins are essential for Shigella virulence and are anchored on the pathogen's surface where they are exposed to the extracellular milieu. We hypothesize that these two proteins represent attractive targets for vaccination. A new class of vaccines that recognize external T3SS components would be expected to provide protection against a broad range of Gram-negative pathogens. To test this hypothesis, the specific aims of the proposed research are to: 1) assess the effects that MxiH- and/or IpaD-specific antibodies have on Shigella virulence-related functions; and 2) directly assess the effectiveness of MxiH and IpaD as vaccines that protect against Shigella infection. The proposed research is in line with the R21 funding mechanism which targets potentially high risk pilot projects whose success would have significant high public health impact and biodefense relevance. The assembled investigative team has the expertise to complete the study as proposed and extend it into a larger and highly significant research program with practical public health implications and biodefense applications. Lay summary: We propose to target the proteins that make up the exposed portions of the type III secretion system to test a new vaccine for the prevention of bacterial dysentery. If successful, a new class of vaccines will be within reach for prevention of infection by many important bacterial pathogens. [unreadable] [unreadable] [unreadable]